Novel enzymes for the degradation of cellulose

Abstract

The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first "extracting" these chains from their crystalline matrix.

Figure 1

Structural overview of a cellulose chain (A) and a simplistic sketch of a Iβ cellulose microfibril (B). Note the simplicity and homogeneity of the cellulose chain. Parallell cellulose chains aggregate into crystalline structures called microfibrils. The arrows indicate the two hydrophobic faces [26] of the microfibril which are thought to be attacking points for cellulases [27].

Figure 2

Structures of CBM33s and GH61s. The figure shows Ta GH61A, a cellulose-active GH61 from Thermoascus aurantiacus (A), CBP21, a chitin-active CBM33 from Serratia marcescens (B), and details of the active sites of these two enzymes (C & D, respectively). Panels A and B show cartoons of the complete proteins; the side chains of two conserved histidines, which are labeled in panels C & D, are also shown. The grey balls in panels C and D represent metal ions (see text for details); the red balls indicate water molecules. Note that the histidines labeled His22 and His28 in panels C and D, respectively, are the N-terminal residues of the mature proteins (i.e. after removal of the signal peptide for secretion) and that the N-terminal amino group participates in coordination of the metal ion.

Figure 3

Summary of the oxidative cleavage of cellulose. In the case of cleavage by CelS2, a CBM33, and Pc GH61D [17] the only oxidized sugars observed are aldonic acids, as indicated in this figure. Other members of the GH61 family seem to generate additional oxidized species, with oxidation at C4 or C6 (see Quinlan et al. [13] and Phillips et al. [16] for further discussion).

Figure 4

HPLC analysis of oxidized products generated by CelS2 and PcGH61D. The main peaks represent aldonic acids of varying chain length (DP, degree of polymerization), as indicated. These soluble products are generated when the same cellulose chain is cut twice by the enzyme, and when the number of sugar units in between the cleavage sites is sufficiently low (longer oligomers are not soluble). The resulting oligomeric products have normal non-reducing ends and are oxidized at the other end. The color coding is as follows: Phosphoric acid swollen cellulose + PcGH61D (black), Avicel + PcGH61D (red), Cellulose nanofibrils + PcGH61D (magenta), and Avicel + CelS2 (blue). Note that the enzymes also produce small amounts of native oligomers [9,17] (not shown in figure). This is most likely the result of a chain being cleaved close to an already existing reducing chain end. It can, however, not be completely excluded that cleavage without oxidation (i.e. normal hydrolysis) occurs under certain conditions. Figure taken from Westereng et al., 2011 [17].

Figure 5

Domain structure of naturally occurring CBM33-containing proteins. Annotations are based on Pfam (http://pfam.sanger.ac.uk) and the number of sequences currently representing each architecture is indicated in brackets. All module families shown are themselves diverse, but have been show experimentally to have (at least) the following substrate preferences: CBM33, chitin, chitosan, cellulose; CBM1, cellulose and chitin; CBM2, chitin, cellulose and xylan; CBM5/2, chitin and cellulose, FnIII, a wide variety of soluble and insoluble substrates; CBM20, granular starch and cyclodextrins; CBM18, chitin; CBM3, cellulose and chitin; CBM14, chitin; PKD (Polycystic kidney disease protein like protein), unknown substrate; LysM, peptidoglycan. Three hydrolytic modules are also present: GH5 (cellulose/mannan/chitosan/xylan and more), GH18 (chitin, chitosan) and GH19 (chitin, chitosan). Note that the CBM33 module almost exclusively occurs at the N-terminus, in accordance with the notion that the N-terminal histidine is crucial for activity (Figure 2).

Figure 6

Artist impression of the interaction between CBP21 and chitin (side view, left; top view, right). The picture highlights how the flat surface of CBP21 fits the flat surface of a β-chitin crystal (the binding interaction is hypothetical and has not been modelled). The surfaces of residues known to interact with chitin [72] are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. Please note that the actual orientation of the enzyme relative to the substrate is unknown; see [18] for an interesting discussion of this topic.

Figure 7

Current view on fungal enzymatic degradation of cellulose. Abbreviations: EG, endoglucanase; CBH, cellobiohydrolase; CDH, cellobiose-dehydrogenase; CBM, carbohydrate-binding module. Note that many cellulolytic enzyme systems have multiple EG and/or CBH that may act on various parts of the substrate, e.g. different crystal faces or parts differing in terms of crystallinity and accessibility. The picture shows a C1 and a C4 oxidizing GH61 which would generate optimal (i.e. non-oxidized) ends for the CBH2 and the CBH1, respectively (oxidized sugars are colored red). Note that the combined action of C1 and C4 oxidizing enzymes may produce native cello-oligosaccharides from the middle of the cellulose chain. The possible consequence of GH61 action is illustrated in the lower left part of the picture, where new attacking points for CBHs are indicated by arrows. CDH may provide GH61s with electrons, but it must be noted that not all organisms have genes encoding for both of these enzyme families in their genome (e.g. Postia placenta has four genes encoding GH61s, but none encoding CDH [81]). Also other non-enzymatic reductants (electron donors) have been demonstrated to induce oxidative activity (e.g. reduced glutathione, ascorbic acid and gallic acid). For more information on the various glucanases and the mechanisms for their synergy, the reader is referred to Kostylev and Wilson, 2012 [43].